High level expression of recombinant human erythropoietin having a modified 5′-UTR

ABSTRACT

The present invention provides an expression construct capable of producing high levels of erythropoietin in mammalian cells. More particularly, the expression construct includes an erythropoietin coding region fused to a unique 5′-UTR sequence and a truncated 3′-UTR. The present invention also provides methods of synthesizing large amounts of erythropoietin and in increasing serum erythropoietin level in individuals in need thereof.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to an expression construct which iscapable of producing high levels of erythropoietin in mammalian cells.More particularly, the present invention relates to an expressionconstruct which includes an erythropoietin coding region fused to aunique 5′-UTR sequence and to the use of such construct for synthesizinglarge amounts of erythropoietin and in increasing serum erythropoietinlevel in individuals in need thereof.

Erythropoietin (EPO) is produced in the adult kidney or the fetal liverin response to tissue hypoxia. Thus, a reduction of tissue oxygen levelresults in upregulation of the EPO gene and in increased levels of EPOin the serum. Increased levels of EPO inhibit apoptosis of the erythroidprogenitor cell in the bone marrow and stimulate its proliferation anddifferentiation which result in a release of erythrocytes into the bloodstream [Krystal, G. (1983) Exp. Hematol. 11, 649-660; Powell J S. et al.(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6465-6469]. On the other hand,lack of EPO in the serum would lead to anemia with fatigue and cellularhypoxia.

Several clinical conditions such as anemia, lung disease or cyanoticheart disease give rise to tissue hypoxia which leads to increasedlevels of serum EPO. However, in patients with renal insufficiency,serum EPO levels remain low in spite of hypoxia. In addition, abnormallylow levels of serum EPO are also seen in anemic patients suffering fromcancer, rheumatoid arthritis, HIV infection, ulcerative colitis, sicklecell anemia and anemia of prematurity.

While the causes of abnormally low levels of EPO include a primarydefect in EPO production in cases of renal disease. and anemia ofprematurity, and a suppression of EPO synthesis by inflammatorycytokines (e.g., IL-1, TNF-α) in cases of certain chronic diseases andcancer, administration of exogenous EPO is expected to circumvent thelow levels of EPO in such conditions.

Indeed, recombinant human EPO (Rhu-EPO) has been successfully used in avariety of clinical conditions to increase production of red bloodcells. Currently, erythropoietin is licensed for use in the treatment ofanemia of renal failure, anemia associated with HIV infection inzidovudine (AZT) treated patients, anemia associated with cancerchemotherapy, myelodysplastic syndromes, prematurity, autologous blooddonation and bone marrow transplantation [Henry, D H. and Spivak, J L.(1995). Curr. Opin. Hematol. 2: 118-124].

However, while EPO administration can benefit a wide variety of patientsits use as a therapeutic agent is limited due to low production yieldsand high production costs.

EPO has been produced from expression vectors using transient and stabletransfections in mammalian cells. These expression vectors included a3.3 kb EPO cDNA under the control of the adenovirus major late promoter[Jacobs K et al. (1985) Nature 313, 806-809], a 5.4 kb long genomic EPO(gEPO) containing the 5′ and 3′ flanking regions transcribed from thesimian virus 40 (SV40) promoter [Lin F K et al. (1985) Proc. Natl. Acad.Sci. U.S.A. 82, 7580-7584], a 2.4 kb ApaI fragment of the EPO genecontaining 58 bp of the 5′ UTR (U.S. Pat. No. 5,688,679 to Powell, J.S., 1986), and a 5′ and 3′ UTR-deleted EPO genomic clone (Park, J. H. etal., Biotechnol. Appl. Biochem. (2000) 32: 167-72). However, thesetransfections resulted in relatively low yields of EPO, which variedbetween 2-7 IU/ml EPO in 48 hours when the transcribed sequence included5′ and 3′ flanking regions, to 56-68 IU/ml in 48 hours when truncated5′-UTR or UTR-deficient EPO constructs were utilized.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, an expression system capable of producing highlevels of EPO devoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided achimeric polynucleotide comprising a nucleic acid sequence encoding anerythropoietin polypeptide attached to a 5′-UTR sequence as set forth bySEQ ID NO:6 or 7.

According to another aspect of the present invention there is provided anucleic acid construct comprising the chimeric polynucleotide.

According to yet another aspect of the present invention there isprovided a eukaryotic cell culture genetically modified to produce atleast 150 international units of erythropoietin per milliliter mediumper 48 hours.

According to still another aspect of the present invention there isprovided a method of increasing blood erythropoietin level in anindividual in need thereof comprising expressing in cells of theindividual a polynucleotide including a nucleic acid sequence encodingan erythropoietin polypeptide attached to a 5′-UTR sequence as set forthby SEQ ID NO:6 or 7 to thereby increase blood erythropoietin level inthe individual.

According to further features in preferred embodiments of the inventiondescribed below, the nucleic acid sequence includes an adenine as partof a guanine-guanine-adenine triplet encoding glycine at position 2 ofSEQ ID NO:10.

According to still further features in the described preferredembodiments the chimeric polynucleotide further includes at least 10 andno more than 15 non-translatable nucleic acids attached to a 3′ end ofthe nucleic acid sequence.

According to still further features in the described preferredembodiments the nucleic acid sequence further includes 12non-translatable nucleic acids attached to a 3′ end of the nucleic acidsequence.

According to still further features in the described preferredembodiments the nucleic acid sequence is set forth by SEQ ID NO:11.

According to still further features in the described preferredembodiments the nucleic acid sequence is set forth by SEQ ID NO:12.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising a promoter fordirecting expression of the chimeric polynucleotide in eukaryotic cells.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising a promoter fordirecting expression of the chimeric polynucleotide in mammalian cells.

According to still further features in the described preferredembodiments the promoter is selected from the group consisting of SV40promoter, CMV promoter, adenovirus major late promoter, Rous sarcomavirus promoter.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising adihydrofolate reductase expression cassette positioned under a controlof a thymidine kinase promoter.

According to still further features in the described preferredembodiments the cells are of a mammalian origin.

According to still further features in the described preferredembodiments the cells are ex-vivo transfected with a mammalianexpression vector including the polynucleotide positioned under thecontrol of a mammalian promoter.

According to still further features in the described preferredembodiments expressing is effected by providing to the individual amammalian expression vector including the polynucleotide positionedunder the control of a mammalian promoter.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing an expression constructwhich is capable of producing high levels of erythropoietin in mammaliancells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a nucleotide sequence of the cloned erythropoietin genomic DNAfragment (SEQ ID NO:8) utilized by the pC2-B3 plasmid. The numberscorrespond to an EPO published genomic sequence (GenBank Accession No.M11319, Lin et al., 1985). Exons appear in uppercase letters whileintrons are in lowercase letters. Coding sequences are underlined, thetranslation start site and stop codon appear in bold letters. Thedeviations from the published sequence (an additional C at position 667,a T to C substitution at position 1445 and a G to A substitution atposition 1775) are marked with bold-italics letters.

FIG. 2 is a schematic illustration depicting assembly of thepASC2M-TKdhfr-8 expression vector. The pASC2M-TKdhfr-8 plasmid wasconstructed by tri-partite ligation of the following fragments: a 5.6 kbfragment containing the pSI vector with the dhfr expression cassette,devoid of the chimeric intron (derived from digestion of pASC2-TKdhfr-10with NheI and SalI), a 1.8 kb fragment containing the 5′ of the EPOgenomic sequence with the minimal 5′ UTR (derived from pASC2-TKdhfr-10digested with NheI and HincII), and a 0.4 kb fragment containing the 3′portion of the EPO genomic sequence with a short 3′ UTR (derived fromthe U1U-C2min3 PCR product digested with HincII and XhoI).

FIG. 3 is a schematic illustration of the pASC2M-TKdhfr EPO expressionvector; functional domains and restriction sites used for constructionare indicated.

FIG. 4 is a nucleotide sequence of the cloned erythropoietin genomic DNA(SEQ ID NO:9) as positioned in the pASC2M-TKdhfr expression vector.Exons appear in uppercase letters while introns are in lowercaseletters. Coding sequences are underlined, translation start site andstop codon appear in italics. The modified nucleotides at the 5′ end arebolded.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a chimeric polynucleotide encoding EPO whichis capable of producing high levels of EPO in mammalian cells and can beused to treat disorders which are associated with, or lead to, abnormalEPO production such as, anemia.

The principles and operation of the chimeric polynucleotide encoding EPOaccording to the present invention may be better understood withreference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Erythropoietin (EPO) is a glycoprotein hormone which regulates theproduction of erythrocytes from the erythroid progenitor cell in thebone marrow. EPO is produced in the adult kidney or the fetal liver inresponse to tissue hypoxia. High levels of EPO in the serum inhibitapoptosis of the erythroid progenitor cell in the bone marrow andpromote its proliferation and differentiation to release erythrocytesinto the blood stream.

Recombinant EPO has been used for reversing anemia caused by disorderssuch as renal failure, HIV infection in zidovudine (AZT) treatedpatients, cancer chemotherapy, myelodysplastic syndromes, prematurity,autologous blood donation and bone marrow transplantation [Henry, D H.and Spivak, J L. (1995). Curr. Opin. Hematol. 2: 118-124].

Previous attempts to produce recombinant human EPO have resulted in pooryields of this commercially important protein.

While reducing the present invention to practice, the present inventorhas constructed a chimeric polynucleotide encoding EPO that produces atleast 150 IU/ml EPO in 48 hours when expressed in mammalian cells, thussubstantially improving synthesis yields as compared to prior art EPOexpression systems.

Thus, according to one aspect of the present invention there is provideda chimeric polynucleotide comprising a nucleic acid sequence encoding anerythropoietin polypeptide attached to a 5′-UTR sequence as set forth bySEQ ID NO:6 or 7.

As used herein, the phrase “chimeric polynucleotide” refers to apolynucleotide sequence which includes sequences from different genes ordifferent species. Accordingly, a chimeric polynucleotide may compriseregulatory sequences and coding sequences that are derived fromdifferent species, or regulatory sequences and coding sequences that arederived from the same species, but are arranged in a manner differentthan that found in nature.

As used herein the term erythropoietin (EPO) polypeptide refers to thehuman EPO protein (GenBank Accession number: NP_(—)000790) which is setforth by SEQ ID NO:10. The EPO polypeptide can be encoded by a naturallyoccurring or synthetic polynucleotide which may or may not includeintronic sequences.

The nucleic acid sequence of the present invention can be acomplementary or genomic DNA fragment which is at least 90%, at least95%, more preferably, 98% or more homologous to the polynucleotidesequence set forth by SEQ ID NO:11 (in the case of cDNA) or 12 (in thecase of a genomic fragment), as determined using the BlastN software ofthe National Center of Biotechnology Information (NCBI) using defaultparameters.

Examples of naturally occurring nucleic acid sequences which encode EPOinclude, but are not limited to, the human EPO coding sequence (GenBankAccession number: NM_(—)000799) or any of its homologues such as rat EPO(GenBank Accession number: NM_(—)017001), mouse EPO (GenBank Accessionnumber: NM_(—)007942) or ox EPO (GenBank Accession number:NM_(—)173909). Synthetic polynucleotide sequences which encode apolypeptide at least 75%, preferably at least 80%, more preferably atleast 85%, most preferably 90%-100% homologous to SEQ ID NO:10 asdetermined by the BlastP software of the National Center ofBiotechnology Information (NCBI) using default parameters can also beused by the present invention to generate EPO.

As is mentioned hereinabove, the chimeric polynucleotide of the presentinvention is designed to express high levels of EPO in mammalian cells.

It will be appreciated that the expression level of a recombinant genein mammalian cells depends on several factors such as transcriptionalcontrol elements, integration position, gene copy number, translationalefficiency, 5′ and 3′ untranslated regions (UTRs) and other factors.

The translational efficiency depends in part on proper splicing, mRNAstabilization (e.g., via capping of the first nucleotide of the RNAtranscript and/or polyadenylation at the 3′-UTR), and initiation oftranslation by the ribosomal complex at the AUG initiation codon.Initiation of translation depends on several features of the sequenceupstream of the main initiation site which is further referred to as aleader sequence or a 5′-UTR sequence, hereinbelow. For example, thenucleotide sequence surrounding the AUG codon (Kozak, M., 1987. NucleicAcids Res. 15: 8125-8148; Kozak, M., 1987. J. Mol. Biol. 196: 947-950;Kozak, M., 1989. Mol. Cell. Biol.9: 5073-5080), the length of the leadersequence (Kozak, M. 1991. Gene. Exp. 1: 117-125; , Kozak, M. 1991. J.Biol. Chem266: 19867-19870), the secondary structure in the leadersequence (Pelletier, J. and Sonenberg, N. 1985. Cell. 40: 515-526 ;Kozak, M. 1986. Proc. Natl. Acad. Sci. USA. 83: 2850-2854; Kozak, M.1989. Mol. Cell. Biol. 9: 5134-5142) and the presence of additional AUGcodons upstream of the main initiation site (Kozak, M., 1989. J. CellBiol. 108: 229-241; Futterer, J. and Hohn, T. 1992. Nucleic Acids Res.20: 3851-3857) can affect the probability of translation initiation at aspecific AUG codon.

Thus, the chimeric polynucleotide of the present invention includes UTRsequences specifically designed capable of improving translationalefficiency of fused EPO coding sequences in eukaryotic cells.

Suitable 5′-UTR sequences for use with the present invention wereselected using theoretical sequence analyses and/or experimental methodsemploying tools such as those available via the Institute of Cytologyand Genetics, The Siberian Branch of the Russian Academy of Science(wwwmgsdotbionetdotnscdotru/mgs/programs/leadermrna/ma_mrna_edothtml).

The result of the above describe analysis is described in Example 1which illustrates a unique UTR sequence (TTTTCTTTTGTTTTGTTTCCACC, SEQ IDNO:6) suitable for use with the present invention.

Another approach for increasing the translation activity of the chimericpolynucleotide of the present invention is via the reduction of theguanine and cytosine content of the sequence following the translationinitiation.

Thus, according to a preferred embodiment of the present invention, atleast one guanine or cytosine nucleotides in the sequence following theAUG initiation codon are replaced with at least one adenine or cytosinenucleotides. It will be appreciated that such a nucleotide change mayresult in an amino acid change in the translated polypeptide unless awobble nucleotide is replaced. Those of skills in the art are capable ofselecting such nucleotide modifications that do not affect the aminoacid sequence of the translated polypeptide.

As is shown in Example 1 of the Examples section which follows a guanineto adenine substitution was introduced at the wobble nucleotide (i.e.,the third guanine nucleotide of the guanine-guanine-guanine triplet)encoding Glycine at position 2 of the EPO polypeptide as set forth bySEQ ID NO:10.

Once selected, the new leader sequence or the modified sequencefollowing the translation initiation codon are incorporated into thechimeric polynucleotide encoding EPO via, for example, site directedmutagenesis.

Methods of introducing site directed mutations are known in the art andinclude, for example, PCR directed mutagenesis. Briefly, a PCR reactionis performed on a DNA template using primers which are selected from theDNA template but include modified, deleted or added nucleotides in orderto direct the PCR reaction to generate a mutated sequence. The PCRconditions are usually adjusted to amplify the template using themodified primers. Such adjustments include for example, increasing theconcentration of the magnesium chloride ions and/or reducing theannealing temperature and the selection of suitable PCR conditions arewithin the capabilities of one skilled in the art.

As was previously shown, production of EPO was increased when the 3′-UTRsequence thereof was truncated (Park, J. H. et al., 2000. Biotechnol.Appl. Biochem. 32: 167-72).

Thus, according to preferred embodiments the chimeric polynucleotide ofthe present invention further includes at least 10 and no more than 15,more preferably, at least 11 and no more than 14, most preferably, 12non-translatable nucleic acids attached to a 3′ end of the nucleic acidsequence encoding EPO.

It will be appreciated that the sequence at the 3′-UTR can be truncatedvia PCR directed mutagenesis as described hereinabove. As is furthershown in Example 1 of the Examples section which follows, using theC2min3 primer (SEQ ID NO:5) the present inventor has shortened the3′-UTR of the polynucleotide encoding EPO to contain only 12 nucleotidesfollowing the TGA translation stop codon.

Altogether, as is shown in Example 2 of the Examples section whichfollows, the modifications introduced into the chimeric polynucleotideencoding EPO of the present invention [i.e., the unique 5′-UTR sequence(SEQ ID NO:6), the guanine to adenine substitution at the wobblenucleotide encoding the Glycine at position 2 of the EPO polypeptide andthe truncated 3′-UTR sequence] resulted in high expression level of EPOin the range of 140-157 IU/ml in 48 hours.

The chimeric polynucleotide of the present invention can be used toproduce the EPO polypeptide via in vitro transcription-translationsystems or preferably by transfecting cells with an expression vectorcontaining the chimeric polynucleotide of the present invention.

According to preferred embodiments the chimeric polynucleotide of thepresent invention is ligated into an expression vector which includes apromoter suitable for directing expression of the EPO polypeptide inmammalian cells.

Constitutive promoters suitable for use with the present invention arepromoter sequences which are active under most environmental conditionsand most types of cells such as the CMV promoter, SV40 promoter,adenovirus major late promoter and Rous sarcoma virus (RSV).

The expression vector of the present invention includes additionalsequences which render this vector suitable for replication andintegration in prokaryotes, eukaryotes, or preferably both (e.g.,shuttle vectors). Typical cloning vectors contain transcription andtranslation initiation sequences (e.g., promoters, enhances) andtranscription and translation terminators (e.g., polyadenylationsignals).

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus (CMV), thelong term repeat from various retroviruses such as murine leukemiavirus, murine or RSV and HIV. See, Enhancers and Eukaryotic Expression,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which isincorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned at approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of EPO mRNA translation. Two distinctsequence elements are required for accurate and efficientpolyadenylation: GU or U rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for the present inventioninclude those derived from SV40.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the recombinantDNA. For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention can further includeadditional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

The expression vector of the present invention can be used to expressthe recombinant EPO polypeptide in mammalian cells (e.g., HeLa cells,Cos cells), yeast cells (e.g., AH109, HHY10, KDY80), insect cells (e.g.,Sf9) or bacteria cells (e.g., JM109, RP437, MM509, SW10).

Preferably, the EPO polypeptide of the present invention is synthesizedby ligating the chimeric polynucleotide (e.g., the polynucleotide setforth by SEQ ID NO:9) into a mammalian, yeast or bacterial expressionvector. Examples of such vectors include but are not limited to thepcDNA3.1, pBK-CMV and pCI vectors which are suitable for use inmammalian cells, the pGBKT7, pLGAD H2-lacZ and pBGM18 vectors which aresuitable for use in yeast cells and the pACK02scKan, pMLBAD, pMLS7vectors which are suitable for use in bacterial cells.

Since the immature human EPO polypeptide (SEQ ID NO:10) is subjected toseveral post-translational modifications including the cleavage of botha 27 amino acid signal peptide at the N-terminal and an arginine residueat the C-terminal, and the addition of three asparagine (N)-linkedtetraantennary oligosaccharides [Dordal M. S., et al. (1985). The roleof carbohydrate in erythropoietin action. Endocrinology. 116: 2293-9;Wasley, L. C., et al. (1991) Blood 77: 2624-2632] the expression vectorof the present invention is preferably constructed for eukaryoticexpression, most preferably, mammalian cell expression.

Examples of mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRepS, D H26S, D HBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Stratagene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Various methods can be used to introduce the expression vector of thepresent invention into mammalian cells. Such methods are generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et al.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods. For example, forstable transfection in dihydrofolate reductase deficient Chinese HamsterOvary (CHO dhfr-) cells the expression vector of the present inventionfurther includes a dihydrofolate reductase expression cassettepositioned under a control of a thymidine kinase promoter.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

Once produced in a host cell, the recombinant EPO protein is secretedinto the culture medium and preferably purified from conditioned mediumusing for example, ion exchange and reverse phase chromatography asdescribed in WO Pat. Appl. No. 86/07594 to Por-Hsiung, L. andStrickland, T.

Purified EPO protein can be further tested for their specific activityusing methods known in the art, including for example determining theincorporation of ⁵⁹Fe into circulating red blood cells of polycythaemicmice which have been exposed to reduced atmospheric pressure,essentially as described elsewhere (European Pharmacopoeia, 4^(th)edition, Directorate for the Quality of Medicines of the Council ofEurope, 2002 pp. 1127-8).

As is mentioned hereinabove, EPO induces proliferation anddifferentiation of the erythroid progenitor cell. Such an effect on theerythroid progenitor cell can be tested by employing the EPO polypeptideof the present invention on methylcellulose bone marrow cultures andscoring the size and number of burst-forming unit-erythroid (BFU-E)colonies as described elsewhere (Martiney, J. A. et al., 2000. Anemia.Infect. Immun. 68: 2259-2267).

The purified EPO polypeptide of the present invention can be used totreat the anemia associated with renal failure, HIV infection inzidovudine (AZT) treated patients, cancer chemotherapy, myelodysplasticsyndromes, prematurity, autologous blood donation and bone marrowtransplantation as reviewed by Henry, D H. and Spivak, J L., 1995(Clinical use of erythropoietin. Curr. Opin. Hematol. 2: 118-124).

Since the chimeric polynucleotide of the present invention is capable ofexpressing EPO in mammalian cells it can be used in in-vivo or ex-vivotherapy of disorders requiring EPO.

Thus, according to another aspect of the present invention there isprovided a method of increasing blood EPO level in an individual in needthereof.

As used herein the phrase “increasing blood EPO level” refers to theproduction of EPO polypeptides in individuals with low EPO levels in theserum which suffer from anemia and/or tissue hypoxia.

According to preferred embodiments of the present invention the methodis effected by expressing in cells of the individual the chimericpolynucleotide of the present invention to thereby increase blood EPOlevel in the individual.

Such expressing can be effected by ex vivo transfection of cells of theindividual with a mammalian expression vector including the chimericpolynucleotide positioned under the control of a mammalian promoter andsubsequent administration of the transfected cells into the blood streamor tissue of the individual.

As is used herein the term “cells” refers to bone marrow cells, stemcells of various cell lineages (e.g., hematopoietic, stromal,epithelial), kidney cells, fetal or adult liver cells, smooth musclecells, endothelial cells which are obtained from the individual via forexample fine needle aspiration or a biopsy. The cells of the presentinvention are isolated from the individual and are optionally culturedunder suitable culturing conditions.

For example, if bone marrow cells are utilized, such cells can beobtained by aspiration from the iliac crest, femora, tibiae, spine, ribor other medullar spaces and low-density marrow cells (<1.077 g/cm³) areisolated by density-cut separation on Ficoll-Hypaque (Pharmacia,Piscataway, N.J.). Progenitor cells are enriched from the low-densitymarrow cells as described elsewhere (Broxmeyer, H. E. et al., 1991, J.Immun., 147: 2586-2594), and are plated at 10⁵ cells/ml in 1%methylcellulose cultures supplemented with suitable growth factors,including, for example, EPO (1 U/ml), recombinant human interleukin-3(rhu IL-3, 100 U/ml, Immunex Corp., Seattle, Wash.) and recombinanthuman steel factor (50 ng/ml, Immunex Corp.). Cultures are maintained ina humidified atmosphere of 5% CO₂ in lowered (5%) oxygen at 37° C.

As is used herein the phrase “ex vivo transfection” refers totransfection of the cells with an expression vector including thechimeric polynucleotide which is designed for expressing EPO in suchcells.

The expression vector described above can be delivered into the cellsusing a variety of delivery approaches, including, but not limited to,microinjection, electroporation, liposomes, iontophoresis orreceptor-mediated endocytosis. The selection of a particular method willdepend, for example, on the cell into which the chimeric polynucleotideis to be introduced.

It will be appreciated that in order to increase blood EPO levels thetransfected cells are administered to the individual in need thereof.

Administration of the cells of the present invention can be effectedusing any suitable route such as intravenous, intra peritoneal, intrahepatic, intra spleenic, intra pancreatic, subcutaneous, transcutaneous,intramuscular, intracutaneous, and/or injection in smooth muscle, usinge.g., a catheter.

The cells of the present invention can be derived from either autologoussources such as self bone marrow cells or from allogeneic sources suchas bone marrow derived from non-autologous sources. Since non-autologouscells are likely to induce an immune reaction when administered to thebody several approaches have been developed to reduce the likelihood ofrejection of non-autologous cells. These include either suppressing therecipient immune system or encapsulating the non-autologous cells ortissues in immunoisolating, semipermeable membranes beforetransplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly (allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002, 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesThechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002, 13: 783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironmrients for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

According to preferred embodiments of the present invention expressingis effected by providing to the individual a mammalian expression vectorincluding the chimeric polynucleotide encoding EPO positioned under thecontrol of a mammalian promoter.

The expression vector including the chimeric polynucleotide encoding EPOcan be administered to the individual per se or as part of apharmaceutical composition which also includes a physiologicallyacceptable carrier. The purpose of a pharmaceutical composition is tofacilitate administration of the active ingredient to an organism.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the expression vectorencoding EPO which is accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (the expression vector including the chimericpolynucleotide encoding EPO of the present invention) effective toprevent, alleviate or ameliorate symptoms of a disorder (e.g., anemiaand/or tissue hypoxia) or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredient are sufficient toprevent anemia and/or tissue hypoxia (minimal effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising the expression vector of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

According to a preferred embodiment of the present invention, theexpression vector including the chimeric polynucleotide encoding EPO ofthe present invention is administered into host tissues using a viralcarrier.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby the present invention will depend on the type transformed. Theability to select suitable vectors according to the cell typetransformed is well within the capabilities of the ordinary skilledartisan and as such no general description of selection consideration isprovided herein.. For example, bone marrow cells can be targeted usingthe human T cell leukemia virus type I (HTLV-I).

Recombinant viral vectors are useful for in vivo expression of thechimeric polynucleotide of the present invention since they offeradvantages such as lateral infection and targeting specificity. Lateralinfection is inherent in the life cycle of, for example, retrovirus andis the process by which a single infected cell produces many progenyvirions that bud off and infect neighboring cells. The result is that alarge area becomes rapidly infected, most of which was not initiallyinfected by the original viral particles. This is in contrast tovertical-type of infection in which the infectious agent spreads onlythrough daughter progeny. Viral vectors can also be produced that areunable to spread laterally. This characteristic can be useful if thedesired purpose is to introduce a specified gene into only a localizednumber of targeted cells.

When retroviruses, for example, are used for polynucleotide transfer,replication competent retroviruses theoretically can develop due torecombination of retroviral vector and viral gene sequences in thepackaging cell line utilized to produce the retroviral vector. Packagingcell lines in which the production of replication competent virus byrecombination has been reduced or eliminated can be used to minimize thelikelihood that a replication competent retrovirus will be produced. Allretroviral vector supernatants used to infect cells are screened forreplication competent virus by standard assays such as PCR and reversetranscriptase assays. Retroviral vectors allow for integration of aheterologous gene into a host cell genome, which allows for the gene tobe passed to daughter cells following cell division.

Mammalian cell systems which utilize recombinant viruses or viralelements to direct expression can be engineered. For example, when usingadenovirus expression vectors, the chimeric polynucleotide encoding EPOcan be ligated to an adenovirus transcription/translation controlcomplex, e.g., the late promoter and tripartite leader sequence.Alternatively, the vaccinia virus 7.5K promoter can be used (Mackett etal., Proc. Natl. Acad. Sci., USA 79:7415-7419, 1982; Mackett et al, J.Virol. 49:857-864, 1984; Panicali et al., Proc. Natl. Acad. Sci., USA79:4927-4931, 1982). Particularly useful are bovine papilloma virusvectors, which can replicate as extrachromosomal elements (Sarver etal., Mol. Cell. Biol. 1:486, 1981). Shortly after entry of this DNA intomouse cells, the plasmid replicates to about 100 to 200 copies per cell.Transcription of the inserted cDNA yielding a high level of expressionmay result without integration of the plasmid into the host cellchromosome. These vectors can be used for stable expression by includinga selectable marker in the plasmid, such as, for example, the neo gene.Alternatively, the retroviral genome can be modified for use as a vectorcapable of introducing and directing the expression of the chimericpolynucleotide encoding EPO in the host cells (Cone and Mulligan, Proc.Natl. Acad. Sci., USA 81:6349-6353, 1984). High level expression canalso be achieved using inducible promoters, including, but not limitedto, the hypoxia-inducible factor (HIF)-I alpha promoter.

Additional expression vectors containing regulatory elements fromeukaryotic viruses such as retroviruses include the SV40 vectors (e.g.,pSVT7 and pMT2), the bovine papilloma virus vectors (e.g., pBV-1MTHA)and vectors derived from Epstein Bar virus (e.g., pHEBO and p205). Otherexemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

It will be appreciated that the expression of the chimericpolynucleotide encoding EPO of the present invention can be specificallyexpressed in blood cells by placing the chimeric polynucleotide sequenceunder the transcriptional control of an erythroid specific promoter suchas the promoter of BVL-1-like VL30 promoter (Staplin W R and Knezetic JA, 2003. BVL-1-like VL30 promoter sustains long-term expression inerythroid progenitor cells. Blood 101: 1798-800).

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes 1-111 Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Construction of a 5′-UTR Modified Erythropoietin ExpressionVector

In order to produce large quantities of erythropoietin (EPO) a chimericpolynucleotide containing a genomic sequence encoding human EPO flankedby a modified 5′-UTR and a 12-nucleotide 3′-UTR was constructed in anexpression vector containing the mouse dihydrofolate reductase (dhfr)expression cassette for stable clone selection in Chinese Hamster Ovary(CHO) cells.

Materials and Experimental Methods

Cell lines—Chinese Hamster Ovary dihydrofolate reductase deficient (CHOdhfr-) cells were maintained in F12 medium (Invitrogen, lifetechnologies, Frederick, Md., USA) supplemented with 10% fetal bovineserum (FBS, Invitrogen, life technologies).

Extraction, manipulation and analysis of DNA—Plasmid DNA was extractedfrom E. Coli cells using the plasmid mini kit (Cat. No. 27106, Qiagen,CA USA) or plasmid midi kit (Cat. No. 12145, Qiagen). DNA restrictionanalysis was performed using restriction enzymes manufactured by NewEngland Biolabs according to the manufacturer's recommendations. DNA waselectrophoresed on Tris-Boric acid-EDTA (TBE) agarose gels and Ethidiumbromide stained gels were documented by the Gel Doc 2000™ geldocumentation system (BioRad, Hercules, Calif. USA) according to themanufacturer's instructions. PCR reactions were performed using a highfidelity enzyme Bio-X-Act (Bio-21049, Bioline, Randolph, Mass. USA) orby Expand high fidelity (Cat. No. 3 300 242, Roche Applied Science,Penzberg, Germany). DNA sequencing was performed using the BigDyeTerminator cycle sequencing kits (Applied Biosystems, Foster City,Calif. USA) according to manufacturer's instruction and extensionreactions were electrophoresed and analyzed on the 377 DNA sequencer(Applied Biosystems) at the Weizmann Institute of Science, Rehovot,Israel.

Experimental Results

Cloning of the human erythropoietin gene—To clone the humanerythropoietin (EPO) gene human male genomic DNA (G1471 Lot: 157888,Promega, Corp., Madison, Wis., USA) was subjected to PCR amplificationusing the high fidelity enzyme Bio-X-Act (Bio-21049, Bioline, Randolph,Mass. USA) and the U1U and U1L PCR primers (Table 1, hereinbelow) whichwere selected from the 5′-UTR and 3′-UTR sequences, respectively, of theEPO coding sequence. The PCR primers were designed to contain the EcoRIand NaeI restriction recognition sites at the 5′ and 3′-ends,respectively. The resultant 2.4 Kb PCR product was subcloned into theEcoRI and EcoR V recognition sites of the pBluescript SK- (Stratagene,La Jolla, Calif.) plasmid which was designated as pC2-B3. Sequencing ofthe pC2-B3 plasmid confirmed the presence of five EPO exons intervenedby four introns and flanked by 61 nucleotides of the 5′-UTR and 220nucleotides of the 3′-UTR. Comparison of the sequence generated from thepC2-B3 plasmid with a published sequence of human erythropoietin(GenBank Accession No. M11319, Lin et al., 1985. Proc. Natl. Acad. Sci.U.S.A. 82: 7580-7584) revealed two polymorphisms in intronic sequences(insertion of C at position 667 and a G→A substitution at position 1775)as well as a T→C substitution at position 1445 which probably reflects aPCR-derived spontaneous mutation in an intronic sequence. Constructionof Erythropoietin/pSI plasmid (pSIC2)—The full-length genomic fragment(2.4 kb) containing the EPO genomic coding sequence was excised frompC2-B3 plasmid using the EcoRI and SalI restriction enzymes and wasfurther ligated into the pSI plasmid (Promega, Madison, Wis. USA) usingthe EcoRI and SalI cloning sites. The resultant EPO clone was designatedpSIC2.

Construction of a modified 5′UTR EPO plasmid (pSC2-2)—To improve thetranslation efficiency of the recombinant EPO a modified 5′ UTR wasdesigned using the translation efficiency algorithm predicting high/lowmRNA expression of a mammalian gene (AV. Kochetov, Institute of Cytologyand Genetics SD RAS, Russia) available throughWwwmgsdotbionetdotnscdotru/mgs/programs/leadermrna/ma_mrna_edothtml. Theselected sequence was introduced into the Syn5 primer (Table 1,hereinbelow) which together with the LPG2 primer which corresponds tonucleotides 1416-1440 at the 3′ end used to amplify the new 5′-UTRregion. In order to increase the translation efficiency the GC contentwas reduced by introducing via the SynS primer a guanine to adenine(G→A) substitution at the third position of the guanine-guanine-guaninetriplet encoding glycine at position 2 of SEQ ID NO:10 (see underlined Ain SEQ ID NO:3, Table 1, hereinbelow). PCR was performed using pSIC2plasmid as template DNA and the resultant PCR product was blunt endedusing T4 DNA polymerase and was digested with XbaI. In parallel, thepSIC2 plasmid DNA was digested with EcoRI, blunt ended and digested withXbaI. The two DNA fragments were ligated and the resultant plasmid wasdesignated as pSC2-2. Further sequence analysis of the pSC2-2 plasmidrevealed the following 5′-UTR sequence: TTTTCTTTTGTTTTGTTTCCACC (SEQ IDNO:6) which is 15-nucleotides shorter at the 5′ end than the 5′-UTRsequence introduced in the Syn5 primer:AATTCTTTTGTTTTGTTTTCTTTTGTTTTGTTTCCACC which is set forth by SEQ IDNO:7. The I 5-nucleotides deletion was generated by either residualdouble strand exonucleolytic activity of the T4 DNA polymerase or by arecombination event occurred in the E. Coli host cell due to thepresence of a highly repetitive sequence. Since the shorter sequence wasstill scored as having a high translation potential using thetranslation predicting algorithm is was selected for further use.

Excision of a chimeric intron in the pSI plasmid—A chimeric intronlocated downstream of the SV40 promoter in the pSI plasmid is designedto improve the expression of cDNAs. Since this intron was assumed to beredundant in expression of genomic sequences containing endogenousintrons it was excised by digestion of the pSC2-2 plasmid with AflIIrestriction enzyme followed by self-ligation. The resultant plasmid wasdesignated pASC2-6.

Insertion of tile TKdhfr into tile EPO expression plasmid(pASC2-10TKdhfr)—A dhfr expression cassette was constructed in theplasmid pRL-TK (E2241 Lot: 158375, Promega) by replacing the LuciferasecDNA with the cDNA of mouse dhfr (Accession No. NM_(—)010049, SEQ IDNO:13). The Luciferase cDNA was excised from the pRL-Tk using the NheIand XbaI restriction enzymes. The cDNA of mouse dhfr previouslysubcloned in the HindIII and BamHI sites of Blue-Script SKII(Stratagene) plasmid was excised using the HindIII and XbaI restrictionenzymes. The NheI and HindIII sticky ends were blunted prior todigestion with the second enzyme. pRL-TK was ligated with the mouse dhfrcDNA fragment and the resultant plasmid was designated pRL-TKdhfr2. Inthe pRL-TKdhfr2 plasmid the mouse dhfr cDNA was cloned downstream of therelatively weak constitutive promoter sequence of HSV-thymidine kinaseand upstream of the SV40 late polyadenylation signal. The dhfrexpression cassette was excised from the pRL-TKdhfr-2 plasmid using theBglII and BamHI restriction enzymes and was cloned into the BamHI siteof the pASC2-6 plasmid. The resultant plasmid was designated:pASC2-TKdhfr-10. The TKdhfr expression cassette was similarly insertedinto the control vector pC2 to generate the control vector pC2-TKdhfr

Modification of 3′UTR—The 3′UTR of the EPO fragment was shortened tocontain only 12 nucleotides downstream of the translation stop codonusing the U1U 5′-primer and the C2min3 modified 3′-UTR primer (Table 1,hereinbelow), which overlaps the translation stop codon. The resultantmodified 3′ sequence was 208 bp shorter than the original 3′ fragment.

TABLE 1 PCR primers Primer name SEQ ID NOs. Forward (F) and reverse (R)primers (5′→3′) U1U SEQ ID NO:1 F: ATGAATTCCCCCGGTGTGGTCACCC U1L SEQ IDNO:2 R: CATGCCGGCCCTCAAGTTGGCCCTG Syn5 SEQ ID NO:3 F:AATTCTTTTGTTTTGTTTTCTTTTGTTTTGTTTCCACCATGGGAGTGCACGGTGAGT LPG2 SEQ IDNO:4 R: ACCCCAAACCAAGTGCCAGGTTCCT C2min3 SEQ ID NO:5 R:TACTCGAGGACACACCTGGTCATCTGTC Table 1: Sequences of PCR primers. BoldedATG (in SEQ ID NO:3) = translation start site; underlined A (in SEQ IDNO:3) = silent Glycine point mutation; bolded TCA (in SEQ ID NO:5) =termination codon;

Assembly of the EPO expression vector—The EPO expression vector(pASC2M-TKdhfr-8) was constructed by tri-partite ligation as is shown inFIG. 2. To reveal the vector fragment the pASC2-TKdhfr-10 plasmid (anintermediate vector containing a pSI plasmid, deletion of the chimericintron, an EPO sequence with a modified 5′ UTR and a dhfr expressioncassette) was digested with the NheI and SalI restriction enzymes. Theresultant 5.6 Kb fragment contained the pSI vector devoid of thechimeric intron, and the dhfr expression cassette. To reveal themodified 5′-UTR fragment the pASC2-TKdhfr-10 was digested with NheI andHincII and a 1.8 kb fragment-as obtained. To reveal the modified 3′ EPOfragment the U1U-min3 PCR product was digested with HincII and XhoI anda 0.4 kb fragment was obtained. Sequencing of the EPO sequence from theresultant pASC2M-TKdhfr-8 expression vector (FIG. 3) confirmed thetri-partite ligation and is presented in FIG. 4.

These results demonstrate the construction of an expression vectorcontaining the EPO genomic sequence with a modified 5′-UTR and a12-nucleotide 3′-UTR sequences and a dhfr expression cassette.

Example 2 CHO Cells Transfected With the 5′-UTR Modified EPO ExpressionVector Express High Level of Erthropoietin

The 5′-UTR modified EPO expression vector was transiently transfected inCHO dhfr- cells and high levels of erythropoietin were secreted into themedium.

Materials and Experimental Methods

Transfections—For DNA transfection 3×10⁶ CHO dhfr- cells were seeded in6-well plates and following one day of incubation were transfected with2.5 μg plasmid DNA in 6 μl Fugene reagent (Roche, Indianapolis, Ind.,USA) according to manufacturer's recommendations. For each construct thetransfection was performed in triplicates.

Immunoassay—The quantity of secreted recombinant EPO was determined inmedia samples collected 48 hours post-transfection using the Quantikine(R&D Systems, Minneapolis, Minn. USA) immunoassay for humanErythropoietin, according to the manufacturer instructions.

Experimental Results

Recombinant EPO with modified 5′-UTR and minimal 3′-UTR region produceshigh levels of EPO in CHO transiently transfected cells—CHO cells weretransiently transfected with the control vector (pC2-TKdhfr, Table 2,hereinbelow) containing an ApaI genomic fragment which contains the EPOcoding sequence (as described in U.S. Pat. No. 5,688,679 to Powell, J.S., 1986) with the pSI expression vector including the dhfr expressioncassette and the EPO genomic coding sequence with modified 5′-UTR andminimal 3′-UTR (pSC2M-TKdhfr, Table 2. hereinbelow) and with thepASC2M-TKdhfr vector (Table 2, hereinbelow) which is similar to thepSC2M-TKdhfr vector however, devoid of the chimeric intron. As is shownin Table 2, hereinbelow, 48 hours post-transfection the level ofsecreted erythropoietin in the media was 140 IU/ml and 157 IU/ml inpSC2M-TKdhfr- and pASC2M-TKdhfr-transfected CHO cells, respectively.

TABLE 2 Production of recombinant EPO by transiently transfected CHOcells % of control Expression Construct IU/ml/48 hours (pC2-TKdhfr)pC2-TKdhfr(control) 98 100 pSC2M-TKdhfr 140 143 pASC2M-TKdhfr 157 161Table 2: The amount of recombinant EPO secreted into the medium by CHOcells transfected with the various expression vectors is presented. IU =International units.

When the expression level of EPO obtained using the teachings of thepresent invention was compared with the expression level obtained usingprior art methods [Powell, J. S., 1986, U.S. Pat. No. 5,688,679 andPark, J. H. et al., Biotechnol. Appl. Biochem. (2000) 32, 167-72] it wasfound that the expression level of EPO in pASC2M-TKdhfr-transfected CHOcells was at least two times higher than the expression level of EPOgenerated using prior art approaches.

Thus, these results demonstrate that the erythropoietin construct of thepresent invention having a modified 5′-UTR and a shorter 3′-UTR iscapable of producing high amounts of erythropoietin which can be usedfor therapeutic and research applications.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A chimeric polynucleotide comprising a nucleic acid sequence encodingan erythropoietin polypeptide attached to a 5′-UTR sequence, whereinsaid 5′-UTR sequence comprises SEQ ID NO:6 or
 7. 2. The chimericpolynucleotide of claim 1, wherein said nucleic acid sequence includesan adenine as part of a guanine-guanine-adenine triplet encoding glycineat position 2 of said erythropoietin polypeptide.
 3. The chimericpolynucleotide of claim 1, wherein said nucleic acid sequence comprisesSEQ ID NO:11.
 4. The chimeric polynucleotide of claim 1, wherein saidnucleic acid sequence comprises SEQ ID NO:12.
 5. The chimericpolynucleotide of claim 1, further comprising a promoter for directingexpression of the chimeric polynucleotide in eukaryotic cells.
 6. Thechimeric polynucleotide of claim 5, further comprising a dihydrofolatereductase expression cassette positioned under a control of a thymidinekinase promoter.
 7. The chimeric polynucleotide of claim 1, furthercomprising a promoter for directing expression of the chimericpolynucleotide in mammalian cells.
 8. The chimeric polynucleotide ofclaim 7, wherein said promoter is selected from the group consisting ofSV40 promoter, CMV promoter, adenovirus major late promoter, and Roussarcoma virus promoter.
 9. An isolated eukaryotic cell comprising thechimeric polynucleotide of claim
 1. 10. The cell of claim 9, which is ofmammalian origin.
 11. The chimeric polynucleotide of claim 1, whereinsaid 5′-UTR sequence consists of SEQ ID NO:6 or 7.